The organic cation transporter 2 regulates dopamine D1 receptor signaling at the Golgi apparatus

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1. 

   Version published to 10.7554/elife.75468 on eLife

   Apr 25, 2022
2. 

   eLife

   Mar 31, 2022

   Author Response:

   Reviewer #1 (Public Review):

   I previously highlighted the need for a physiologically relevant cell type, the issue of showing that OCT2 is not only sufficient but also required for activation of Golgi-localized receptor, and a concern about how cytoplasmic dopamine gains access to the Golgi lumen. While the latter concern somewhat remains, this version satisfactorily addresses these issues and I believe the study will be of interest to a large audience.

   We are grateful for the positive comments and that the reviewer deems this version of the manuscript as improved. To address the remaining concern of the reviewer, we have now added data (Supplementary Figure 5d and 7d) to show endogenous OCT2 localization at the Golgi in HeLa cells and MSNs. Importantly, we have confirmed the specificity of our OCT2 antibody by …

   Author Response:

   Reviewer #1 (Public Review):

   I previously highlighted the need for a physiologically relevant cell type, the issue of showing that OCT2 is not only sufficient but also required for activation of Golgi-localized receptor, and a concern about how cytoplasmic dopamine gains access to the Golgi lumen. While the latter concern somewhat remains, this version satisfactorily addresses these issues and I believe the study will be of interest to a large audience.

   We are grateful for the positive comments and that the reviewer deems this version of the manuscript as improved. To address the remaining concern of the reviewer, we have now added data (Supplementary Figure 5d and 7d) to show endogenous OCT2 localization at the Golgi in HeLa cells and MSNs. Importantly, we have confirmed the specificity of our OCT2 antibody by showing that immunostaining is abrogated in cells expressing OCT2 shRNAs (Supplementary Figure 5d).

   Reviewer #2 (Public Review):

   Weaknesses

   The selectivity of the transport inhibitors is overstated. Corticosterone is described as an OCT3 inhibitor when it also inhibits OCT2. Imipramine is described as an OCT2 inhibitor when it also inhibits OCT1, OCT3, and the plasma membrane monoamine transporter (PMAT). Given that only OCT2 expression is quantified in any of the cells under study, a clear description of the relative potencies of these inhibitors at the other transporters is necessary to justify the definitive conclusions the authors make about the exclusive role of OCT2.

   We have now added additional data to describe the exclusive role of OCT2 in regulating D1DR signaling at the Golgi. Our previous report demonstrated that HeLa cells also express OCT3 and 10M corticosterone inhibits epinephrine-mediated activation of the Golgi-localized 1AR (PMID: 28553949). We acknowledge that corticosterone has been reported to also inhibit OCT2 uptake of DA in a stable transfection system with a Ki Value of 500nM (PMID: 9812985). However, in our hands, we did not observe inhibition of D1DR signaling at the Golgi when cells were treated with 10M corticosterone (Figure 2b). To bolster the validity of our conclusions and the specificity of OCT2 in transporting dopamine in HeLa cells, we have included additional data using two different shRNAs against OCT2 to show that genetic knock-down of OCT2 in HeLa cells blocks D1DR activation at the Golgi. By contrast, control (scrambled) shRNA had no effect on D1DR activation at the Golgi, suggesting specificity of OCT2 shRNA transfection in HeLa cells (Supplementary Figure 6b). Importantly, SKF81297, a membrane permeant agonist that diffuses across the membrane and does not require OCT2, can still reach the Golgi membranes and activate D1DR at the Golgi even when OCT2 is genetically knocked down (Figure 2 d-g).

   The authors cite work demonstrating OCT localization to intracellular membranes, including nuclear and Golgi membranes. This work focused exclusively on OCT3. This must be clearly stated.

   Thank you for pointing out this important point. We have now clarified this in the main text. Additionally, we have provided new data showing OCT2 localization on the plasma membrane and the Golgi in HeLa and MSNs (supplementary Figure 5d and Supplementary Figure 7d). We have confirmed the specificity of the antibody using shRNA against OCT2 (Figure 2e and Supplementary Figure 5d).

   There are instances in which the conclusions made by the authors are not fully justified by the data. The authors state that OCT2 expression is "negligible" in hippocampal tissue, but there is a clear OCT2-immunoreactive band in the western blot. They state that HEK293T cells "do not express OCT2", but there is a clear OCT2-immunoreactive band in their western blot.

   We agree. We have revised the writing of the main text to clarify that OCT2 expression is lower in hippocampal tissue and HEK293 cells compared to Striatal and HeLa cells.

   Also, regarding the western blot data: The authors describe primary murine striatal MSNs where, "OCT2 is expressed at high levels." The data they refer to describe OCT2 expression in bulk striatal tissue which, while it does include MSNs, also includes other neuronal types, glial cells, and vascular tissue. There was no specific measurement of OCT2 expression specifically in MSNs, so the statement overstates the findings of the western blot.

   We have now added immunostaining of OCT2 in isolated striatal MSNs, digested from striatum tissue, mechanically separated and passed through a 40m cell strainer to eliminate vascular tissues. Remaining cells were plated using neural basal media. Cultures were then treated with 2μM Cytosine arabinosine (Sigma-Aldrich) at day 3 to inhibit glial cells growth. As a result, we did have less non-neuronal cells in the culture. The representative image in Supplementary Figure 7d shows OCT2 expression on both the plasma membrane and the Golgi in MSN. We have confirmed the specificity of this OCT2 antibody using shRNA against OCT2 (Supplementary Figure 5d).

   Reviewer #3 (Public Review):

   The main weakness is the physiological relevance of the observation. As it stands, how much dopamine gets into the Golgi and whether it activates endogenous D1DR are not clear.

   The authors use 10µM dopamine in some assays and 10nM in others, which complicates the interpretation. Gründemann 1998, referenced by the authors, have provided rates of transport of dopamine by OCT2, which the authors could use to estimate how much dopamine will get into the Golgi over time. The authors can match a dose response of dopamine to these estimates. This is also important as Nb6B9 recruitment of HEK293 cells seem to increase over ~1000 sec, which is comparable to Nb80 recruitment to B1AR by norepinephrine (~20 min).

   We have added new data to quantify Golgi-D1DR activation in response to various doses of dopamine. Increased concentrations of dopamine were added to the same cells over time and Nb6B9 or miniGs recruitments to the plasma membrane and the Golgi were quantified. Nb6B9 recruitment quantifications are shown in Figure 1c and supplementary Figure 1b for the Golgi and the plasma membrane, respectively. MiniGs recruitment quantifications are shown in supplementary Figure 3b. We were able to detect plasma membrane activation of D1DR starting at 10nM dopamine concentration using both biosensors. Subtle D1DR activation at the Golgi were detected at 10nM and 100nM dopamine by miniGs and Nb6B9, respectively. Although we do not have exact measurements for Nb6B9 versus miniGs binding affinities to activated D1DR, these observations suggest that miniGs is more sensitive in detecting activated D1DR. It is important to clarify that our ability to precisely measure D1DR activation by low concentrations of dopamine at the Golgi is limited due to higher cytoplasmic background of biosensors that mask their earlier recruitment to the Golgi. Thus, to better quantify the increase in fluorescence intensity at the Golgi after addition of agonist, values were normalized to the baseline following each dose of agonist addition. Each baseline value was set to 1 to measure the fold change in fluorescence. These calculations were done by Microsoft Excel. At 10nM agonist concentration, we first observe the plasma membrane recruitment of the biosensors. As a result, when calculating fluorescence intensity of the biosensor recruitment to the Golgi, we initially see a decrease in the cytoplasmic fluorescence due to biosensor recruitment to the plasma membrane, followed by an increase recruitment to the Golgi (Figure 1c, Supplementary Figure 1c and Supplementary Figure 3b).

   Based on the calculated rate constant for OCT2 in vivo and the known water space of average cells, cytoplasmic concentration of DA at equilibrium were calculated to be ~ 10-fold higher than the extracellular concentration (PMID: 9812985). For instance, Grundemann et al have shown that addition of 100nM DA in the extracellular environment of OCT2 expressing cells results in the accumulation of 4 pmol/mg in cells after 10min. Considering the average weight of a cell to be ~ 1ng, this translates into: 4x10^-12 / 10^6 = 4x10^-18 mol/cell.

   Given the average volume of a cells is ~ 4pL, thus in 10 mins, we end up getting: 4x10^-18/4x10^-12= 1x10^6 M= 1M dopamine in the cytoplasm, which is 10-fold higher than the added extracellular concentration.

   In our measurements, we were able to detect activation of the Golgi-localized D1DRs even at low concentrations of exogenously added DA (10nM) (Supplementary Figure 3).

   Another concern is the specificity of some of the reagents used. For example, a high dose of imipramine is used to block OCT2. Imipramine acts on many other targets, including monoamine uptake and D2 dopamine receptor. Genetically depleting OCT2 in neurons, or at least in HeLa cells, is critical to show that OCT2 is required for Golgi activation.

   We have now included additional data showing that lower concentration of imipramine (10M) also inhibits D1DR activation at the Golgi (Figure 2b). Additionally, we have added data describing that genetic knock-down of OCT2 in HeLa cells, using two different shRNAs that we have validated, abrogates Golgi-localized D1DR signaling (Figure 2 and 3), highlighting the specificity of OCT2 in this signaling regulation.

   Repurposing Nb6B9 to detect D1DR is clever. But it also raises concerns about the specificity of the Nb6B9. Does it bind other catecholamine receptors that are in neurons, which could be cross-activated by dopamine? Further, Nb6B9 was originally designed to stabilize an active form of the receptor. The effects could be a due to Nb6B9 expression stabilizing active D1DR.

   We agree that these conformational sensitive nanobodies stabilize an active form of receptor but when they are expressed at high concentrations. In order to function as biosensors, we express them at a very low concentration. We have calculated this previously when we first established the use of nanobody-based biosensors (Nb80) to be around 10nM in cells (PMID: 23515162). Importantly, the nanobody-based biosensor’s localization (Nb6B9-GFP) is diffused in the cytoplasm and not bound to the receptor in the absence of the agonist (Figure 1b top panel). This further confirms that at this low concentration it does not stabilize an active form of the receptor. Only after addition of dopamine, Nb6B9 can bind to the receptor with high affinity and is recruited first to the plasma membrane and then the Golgi membranes (Figure 1b). This can be better appreciated in Supplementary movies 1-3.

   Additionally, all of the previously reported nanobody-based biosensors and miniG proteins (PMID: 23515162, PMID: 28553949, PMID: 29754753, PMID: 29523687, PMID: 31263273) have relied on conditions where target receptors are over-expressed. This is because endogenous GPCRs are expressed at very low level and the cytoplasmic presence of nanobody or miniG-based biosensors presents a high level of background. Thus, in order to achieve a higher signal-to-noise ratio and to increase the level of detection, receptor expression has been increased which is why we do overexpress D1DR in MSNs. Therefore, we do not believe that the expression of other catecholamine receptors which are endogenously expressed at low levels in neurons could be a reason why Nb6B9 is recruited to D1DRs.

   The miniGs and Nb37 experiments in Supplemental Figures 3 and 4 are also important in this regard, but they are not convincing. Nb37 and miniGs shows much weaker recruitment, which suggests that Nb6B9 might be changing receptor sensitivity. A dose response will help here. Also, it is critical to know how recruitment of all these sensors compare to positive control (e.g., B1AR with NE) and negative control (e.g., opioid receptors) for this experiment and for Fig 1.

   As mentioned earlier, we have now added additional data measuring dose response recruitment of our biosensor to D1DR, 1AR (a positive control) and an opioid receptor (a negative control) (Figure 1c and Supplementary Figure 1b-d and supplementary Figure 3b). Detection of 1AR activation at the plasma membrane and the Golgi by Nb6B9 occurred at comparable epinephrine concentrations (Supplementary Figure 1c and d). Importantly, Nb6B9 was unable to detect activation of Gi-coupled GPCRs such as delta opioid receptors (Supplementary Figure 1e), indicating its specificity of binding to catecholamine receptors where Nb6B9 binding sites are conserved (Supplementary Figure 1a). These dose-dependent responses were observed with over-expression of specific receptor and only upon addition of their specific ligands.

   MiniG_s recruitment quantifications are shown in supplementary Figure 3b. We were able to detect plasma membrane activation of D1DR starting at 10nM dopamine concentration using both Nb6B9 and MiniGs biosensors. Subtle D1DR activation at the Golgi were detected at 10nM and 100nM dopamine by miniGs and Nb6B9, respectively. Although we do not have exact measurements of Nb6B9 versus miniGs binding affinities to activated D1DR, these observations suggest that miniGs is, in fact, more sensitive in detecting activated D1DR.

   The high cytoplasmic background of biosensors prevents easy visualization of biosensor recruitment to any membranes, including the plasma membrane by endogenously expressed GPCRs. Thus, in order to achieve a higher signal-to-noise ratio and to increase the level of detection, receptor expression has been increased. While the endogenous receptor activity can potentially be monitored by employing super resolution microscopy techniques such as TIRF microscopy to track single-particles of photo-switchable nanobodies (PMID: 29045395, PMID: 33214152), this type of TIRF microscopy is only suitable for cell surface receptors, and not applicable to receptors located at internal membrane locations such as the Golgi.

   Nb37 shows much weaker recruitment to D1DR/Gs receptors compared to NB6B9 even at higher DA concentration (10M). This is expected and consistent with measured binding affinities of the two biosensors. We have previously measured the binding affinity of Nb37 to agonist bound 2AR/Gs complex to be ~800nM. By comparison, binding affinities of Nb80 and Nb6B9 (receptor nanobodies) for agonist bound 2AR have been reported at ~10nM (PMID: 23515162, PMID: 24056936). Although we do not have the exact affinity measurements for Nb6B9 and Nb37 for D1DR, our dose dependent recruitment experiments suggest comparable binding affinities of these biosensors to the D1DR/Gs complex as those reported for the 2AR/Gs complex (Figure 1c and Supplementary Figure b-d). To improve visualization of the weak Nb37 signal, we fixed and permeabilized MSNs and immuno-stained them with GFP antibodies. Our new data in Figure 3c shows Nb37-GFP recruitment to activated D1DR in MSNs after 5 min of 10M DA stimulation, providing evidence for functional G protein coupling of D1DR in physiologically relevant cell type.

   The localization of the endogenous D1DR in the Golgi of striatal neurons and the activation by DA is a critical part of the paper. However, it is missing controls. GPCR antibodies are notoriously bad, and this commercial D1DR antibody is recommended only for immunoblotting. The authors need to confirm that this antibody is specific.

   To test the specificity of D1DR antibody used to detect endogenous D1DR localization in MSNs, we used a commercially available D1DR antibody that has been validated by immunostaining. Using this new antibody, we were able to detect endogenous D1DR on both the plasma membrane and the Golgi membranes in MSNs (Supplementary Figure 7c). Importantly, D1DR immunostaining was largely diminished when MSNs were immuno-stained in the presence of D1DR blocking peptide (Supplementary Figure 7c). Of course, we use the same laser power and exposure time to image our samples using a spinning disk microscope.

   Read the original source
3. 

   eLife

   Mar 31, 2022

   Evaluation Summary:

   This study uses a tour de force of biosensor constructs providing evidence that dopamine transport by OCT2 across the plasma membrane and also (presumably) into the Golgi activates GPCR signaling at the Golgi leading to cAMP production and PKA activation. Thus, intracellularly compartmentalized signaling underlies aspects of Dopamine D1 receptor signaling. The work will be of interest to scientists working on the cell biology of dopamine signaling. While the data support the model overall, there are concerns that need to be addressed including specificity of the reagents used and the actual intracellular localization of D1DR and OCT2.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. …

   Evaluation Summary:

   This study uses a tour de force of biosensor constructs providing evidence that dopamine transport by OCT2 across the plasma membrane and also (presumably) into the Golgi activates GPCR signaling at the Golgi leading to cAMP production and PKA activation. Thus, intracellularly compartmentalized signaling underlies aspects of Dopamine D1 receptor signaling. The work will be of interest to scientists working on the cell biology of dopamine signaling. While the data support the model overall, there are concerns that need to be addressed including specificity of the reagents used and the actual intracellular localization of D1DR and OCT2.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

   Read the original source
4. 

   eLife

   Mar 31, 2022

   Reviewer #1 (Public Review):

   I previously highlighted the need for a physiologically relevant cell type, the issue of showing that OCT2 is not only sufficient but also required for activation of Golgi-localized receptor, and a concern about how cytoplasmic dopamine gains access to the Golgi lumen. While the latter concern somewhat remains, this version satisfactorily addresses these issues and I believe the study will be of interest to a large audience.

   Read the original source
5. 

   eLife

   Mar 31, 2022

   Reviewer #2 (Public Review):

   Puri, Romano, et al. investigated the possibility that, as they and others have demonstrated for adrenergic receptors, functional dopamine D1 receptors can be localized to and activated at Golgi membranes. They use an impressive array of genetically encoded biosensors to visualize the cellular localization of dopamine receptors, their activation, and downstream signaling events. They find that bath application of dopamine, a membrane impermeant ligand for D1 receptors, causes activation of Golgi-localized D1 receptors in HeLa cells and primary cultured striatal neurons, but not in HEK293 cells. They find that dopamine activation of Golgi-localized D1 receptors requires expression of OCT2, a transmembrane catecholamine transporter. In HEK cells, which they show express low levels of OCT2, Golgi-localized D1 …

   Reviewer #2 (Public Review):

   Puri, Romano, et al. investigated the possibility that, as they and others have demonstrated for adrenergic receptors, functional dopamine D1 receptors can be localized to and activated at Golgi membranes. They use an impressive array of genetically encoded biosensors to visualize the cellular localization of dopamine receptors, their activation, and downstream signaling events. They find that bath application of dopamine, a membrane impermeant ligand for D1 receptors, causes activation of Golgi-localized D1 receptors in HeLa cells and primary cultured striatal neurons, but not in HEK293 cells. They find that dopamine activation of Golgi-localized D1 receptors requires expression of OCT2, a transmembrane catecholamine transporter. In HEK cells, which they show express low levels of OCT2, Golgi-localized D1 receptors can be activated by a membrane-permeant D1 receptor agonist, leading to apparent activation of PKA and contributing to D1-induced increases in cAMP concentration. These findings add to the growing body of evidence that G-protein-coupled receptors for monoamines can be localized to intracellular membranes and activated by their endogenous ligands in a transporter-gated fashion. Intracellular D1 receptors represent an intriguing novel mechanism by which dopamine may influence neuronal activity, and which may contribute to the physiological and behavioral actions of this catecholamine transmitter.

   The conclusions of this manuscript are generally supported by the data. The authors have demonstrated that D1 receptors can be localized to, and activated at, the Golgi apparatus, and that activation of these intracellular receptors requires transmembrane catecholamine transporters. Overall, the work contributes to the growing body of evidence that G-protein-coupled receptors can be activated not only at the plasma membrane, but also from intracellular membranes. This has been demonstrated repeatedly for beta-adrenergic receptors but has never been demonstrated for dopamine receptors. While the authors do not describe physiological consequences of Golgi D1 activation in striatal neurons, the demonstration that they occur and are activated in striatal neurons has profound implications for our understanding of the mechanisms by which dopamine and drugs of abuse influence striatal function.

   Strengths:

   The use of multiple genetically-encoded fluorescent sensors has allowed the authors to build a strong base of evidence that dopamine D1 receptors can be localized to the Golgi and that they can be shifted to their active conformation by D1 ligands. They also provide evidence that activation of Golgi-localized receptors contributes to D1-induced increases in cAMP and leads to activation of PKA. Thus, Golgi D1 receptors appear to be fully functional Gs-coupled receptors.

   The authors make very effective use of the differences in Golgi D1 activation between HeLa and HEK cells to illustrate the potential role of OCT2 in dopamine-induced activation of intracellular receptors. They make effective use of OCT inhibitors and genetic overexpression of OCT2 to demonstrate that dopamine activation of Golgi D1 receptors requires the expression of this catecholamine transporter. The further use of the membrane permeant D1 agonist both confirms the identity of the receptors and demonstrates the necessity of transporter expression and function for dopamine-induced activation.

   The authors have provided evidence that Golgi-localized D1 receptors also occur in cultured mouse striatal neurons, which suggests that this signaling system may contribute to the powerful actions of dopamine in these neurons. This would represent a highly novel potential mechanism underlying dopamine-induced regulation of neuronal physiology and behavior. The authors demonstrate, using immunofluorescence that endogenous D1 receptors are localized to Golgi in cultured neurons. It is unclear why, in their subsequent imaging experiments, they used exogenously expressed SNAP-GFP tagged D1 receptors.

   Weaknesses:
   Most of the data are from cell lines exogenously expressing D1 receptors. The Golgi localization and OCT2-dependent activation are demonstrated in cultured murine striatal neurons, but downstream signaling or physiological responses are only described in HeLa and/or HEK cells.

   The selectivity of the transport inhibitors is overstated. Corticosterone is described as an OCT3 inhibitor when it also inhibits OCT2. Imipramine is described as an OCT2 inhibitor when it also inhibits OCT1, OCT3, and the plasma membrane monoamine transporter (PMAT). Given that only OCT2 expression is quantified in any of the cells under study, a clear description of the relative potencies of these inhibitors at the other transporters is necessary to justify the definitive conclusions the authors make about the exclusive role of OCT2. The authors cite work demonstrating OCT localization to intracellular membranes, including nuclear and Golgi membranes. This work focused exclusively on OCT3. This must be clearly stated.

   There are instances in which the conclusions made by the authors are not fully justified by the data. The authors state that OCT2 expression is "negligible" in hippocampal tissue, but there is a clear OCT2-immunoreactive band in the western blot. They state that HEK293T cells "do not express OCT2", but there is a clear OCT2-immunoreactive band in their western blot. Also regarding the western blot data: The authors describe primary murine striatal MSNs where, "OCT2 is expressed at high levels." The data they refer to describe OCT2 expression in bulk striatal tissue which, while it does include MSNs, also includes other neuronal types, glial cells, and vascular tissue.

   There was no specific measurement of OCT2 expression specifically in MSNs, so the statement overstates the findings of the western blot.

   Read the original source
6. 

   eLife

   Mar 31, 2022

   Reviewer #3 (Public Review):

   This study repurposes Nb6B9, a nanobody originally used to stabilize adrenergic receptors in an active conformation, to detect active D1 Dopamine Receptors at different regions of the cell. Dopamine is known to be transported by the OCT2 transporter. The authors observe that HeLa cells and cultured primary neurons which have endogenous OCT2, recruit Nb6B9 to exogenously expressed D1DR in the Golgi, but HEK293 cells which do not have endogenous OCT2 do not. HeLa cells also recruit other biosensors that read out activated Gs protein and PKA. Expressing OCT2 in HEK293 cells causes recruitment of Nb6B9 in response to dopamine.

   GPCR signaling from Golgi is an emerging phenomenon that is still not well understood. Recent studies have reported Golgi activation of adrenergic receptors by norepinephrine and opioid …

   Reviewer #3 (Public Review):

   This study repurposes Nb6B9, a nanobody originally used to stabilize adrenergic receptors in an active conformation, to detect active D1 Dopamine Receptors at different regions of the cell. Dopamine is known to be transported by the OCT2 transporter. The authors observe that HeLa cells and cultured primary neurons which have endogenous OCT2, recruit Nb6B9 to exogenously expressed D1DR in the Golgi, but HEK293 cells which do not have endogenous OCT2 do not. HeLa cells also recruit other biosensors that read out activated Gs protein and PKA. Expressing OCT2 in HEK293 cells causes recruitment of Nb6B9 in response to dopamine.

   GPCR signaling from Golgi is an emerging phenomenon that is still not well understood. Recent studies have reported Golgi activation of adrenergic receptors by norepinephrine and opioid receptors by synthetic opioids. The main advance of this study is that it adds dopamine receptors to this list. As with these previous papers, this study takes advantage of biosensors that are still relatively new in the field.

   The main weakness is the physiological relevance of the observation. As it stands, how much dopamine gets into the Golgi and whether it activates endogenous D1DR are not clear.

   The authors use 10µM dopamine in some assays and 10nM in others, which complicates the interpretation. Gründemann 1998, referenced by the authors, have provided rates of transport of dopamine by OCT2, which the authors could use to estimate how much dopamine will get into the Golgi over time. The authors can match a dose response of dopamine to these estimates. This is also important as Nb6B9 recruitment of HEK293 cells seem to increase over ~1000 sec, which is comparable to Nb80 recruitment to B1AR by norepinephrine (~20 min).

   Another concern is the specificity of some of the reagents used. For example, a high dose of imipramine is used to block OCT2. Imipramine acts on many other targets, including monoamine uptake and D2 dopamine receptor. Genetically depleting OCT2 in neurons, or at least in HeLa cells, is critical to show that OCT2 is required for Golgi activation.

   Repurposing Nb6B9 to detect D1DR is clever. But it also raises concerns about the specificity of the Nb6B9. Does it bind other catecholamine receptors that are in neurons, which could be cross-activated by dopamine? Further, Nb6B9 was originally designed to stabilize an active form of the receptor. The effects could be a due to Nb6B9 expression stabilizing active D1DR.

   The miniGs and Nb37 experiments in Supplemental Figures 3 and 4 are also important in this regard, but they are not convincing. Nb37 and miniGs shows much weaker recruitment, which suggests that Nb6B9 might be changing receptor sensitivity. A dose response will help here. Also, it is critical to know how recruitment of all these sensors compare to positive control (e.g., B1AR with NE) and negative control (e.g., opioid receptors) for this experiment and for Fig 1.

   The localization of the endogenous D1DR in the Golgi of striatal neurons and the activation by DA is a critical part of the paper. However it is missing controls. GPCR antibodies are notoriously bad, and this commercial D1DR antibody is recommended only for immunoblotting. The authors need to confirm that this antibody is specific.

   Overall, this is an interesting paper that presents a new observation that D1DR could be activated on the Golgi in neurons. The paper will be significantly strengthened by improving the physiological relevance and being rigorous in providing controls.

   Read the original source
7. 

   Version published to 10.1101/2020.11.23.394445v3 on bioRxiv

   Nov 15, 2021
8. 

   Version published to 10.1101/2020.11.23.394445v2 on bioRxiv

   Feb 6, 2021
9. 

   Version published to 10.1101/2020.11.23.394445v1 on bioRxiv

   Nov 23, 2020